A Multidimensional Diversity‐Oriented Synthesis Strategy for Structurally Diverse and Complex Macrocycles

Abstract Synthetic macrocycles are an attractive area in drug discovery. However, their use has been hindered by a lack of versatile platforms for the generation of structurally (and thus shape) diverse macrocycle libraries. Herein, we describe a new concept in library synthesis, termed multidimensional diversity‐oriented synthesis, and its application towards macrocycles. This enabled the step‐efficient generation of a library of 45 novel, structurally diverse, and highly‐functionalized macrocycles based around a broad range of scaffolds and incorporating a wide variety of biologically relevant structural motifs. The synthesis strategy exploited the diverse reactivity of aza‐ylides and imines, and featured eight different macrocyclization methods, two of which were novel. Computational analyses reveal a broad coverage of molecular shape space by the library and provides insight into how the various diversity‐generating steps of the synthesis strategy impact on molecular shape.


General details
• Macrocycles derived from 21a-d are shown in Scheme S1.
• The optimization of the Pauson-Khand Macrocyclization Reaction is shown in Table S1.
• A list of all azide alkyne cycloaddition macrocyclisations (apart from macrocycles derived from 21a-d), are shown in Table S2, and other macrocyclisations are shown in Table S3.
• The aim of the macrocyclic library was to develop a strategy to synthesise rings ranging from 15-to 33-membered rings. This strategy could be applied to smaller 12-to 14-membered macrocycles with the use of different building blocks and chemistry. We are currently working towards the synthesis of a library of smaller natural product like macrocyclic rings.
• Another limitation of our current chemistry was the synthesis of 1,5-triazoles with an amine present in the linear precursor. Attempted RuAAC macrocyclisation coupling conditions of the linear precursors featuring amine linkers resulted in decomposition of the starting material, and as such was not pursued.
• The yield of the macrocyclisation can vary depending on the method used. In most cases each macrocyclisation method was only used in one example therefore we cannot correlate this with substrate dependence. In the case of the Huisgen cycloaddition macrocyclisation reactions the yields were relatively consistent. Further work will be conducted to investigate the effect of substrate dependence on each macrocyclisation method used.

Analysis of comparative PMI plot
The synthetic drug (non-macrocyclic) collection predominately contains compounds with rod-like shapes with some disk-like features. The natural products and macrocyclic-based compound collections span a significantly wider range of molecular shape space, from sp 2 features extending into spherical features. The synthesised macrocyclic DOS library also displays a high level of S45 molecular shape diversity and covers a molecular shape space comparatively as broad as natural products, overlapping to a substantial extent with the drug collection as well as the macrocycles in clinical development.

Top selling drugs
Lipitor 60 randomly selected natural products

Rifamycin B
O1c2c3c4c ( Bleomycin A2 INO 4885    Top contributing parameters to each principal component are marked in grey, the darker grey, the more contribution in each column. The values were normalised automatically by the MOE software.

General experimental methods
All non-aqueous reactions were carried out under nitrogen or argon with dry and freshly distilled solvents using oven-dried glassware unless otherwise stated. Room temperature (rt) refers to ambient temperature. A temperature of 0 °C was maintained using an ice-water bath. A temperature of -78 °C was maintained using an acetone-dry ice bath. Reactions under microwave heating were performed in sealed vials using a CEM Discover SP microwave reactor.
All reagents and solvents were used as obtained from commercial sources unless otherwise stated. Yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated.
Reactions were monitored using thin layer chromatography (TLC) or low resolution mass spectra (LRMS). Yields around 100% were recorded as quantitative (quant. Optical rotations were recorded on a Perkin Elmer 343 polarimeter. [α] T D values are reported in 10 -1 degcm 2 g -1 at 589 nm, concentration (c) is given in gdL -1 .
Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum One (FT-IR) spectrometer with internal referencing as neat films. Selected absorption maxima (ν max ) are reported in wavenumbers (cm -1 ) and the following abbreviations are used: w, weak; m, medium; s, strong; br, broad.
In proton magnetic resonance spectra (

GSP-1 Synthesis of azido compounds via diazonium salt
The aromatic amine (1.0 eq) was suspended or dissolved in water. Concentrated sulfuric acid (4 mL) and NaNO 2 (1.05 eq) were added at 0 °C. The reaction mixture was stirred at 0 °C for 20 min and a solution of NaN 3 (1.05 eq) dissolved in water at 0 °C added dropwise. Then EtOAc was added and the two layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO 4 , filtered and concentrated.
After stirring at rt overnight, the mixture was washed with 5% aq. HCl, sat. aq. NaHCO 3 and brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was either purified by FCC or used without further purification. The (crude) product was dissolved in anhydrous DMF (30 mL) and NaN 3 (1.2 eq) added. After stirring at rt overnight, the mixture was partitioned between Et 2 O and water. The organic phase was washed with sat. aq. NaHCO 3 and brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude product was purified by FCC.

GSP-3 Synthesis of the aza-ylides
Procedure a) the azido building block (1.0 eq) was dissolved in THF with 4 Å molecular sieves. PBu 3 (1.0 eq) was added. The reaction mixture was stirred at room temperature until TLC indicated complete turnover (typically 1-2 h). If starting material was not completely consumed, additional PBu 3 was added in portions (typically 0.05 eq) until TLC indicated complete turnover. This aza-ylide was used as a solution.
Procedure b) the azido building block (1.0 eq) and PPh 3 (1.0 eq) were dissolved in THF with 4 Å molecular sieves. The reaction mixture was stirred at room temperature until TLC indicated complete turnover (typically 3-4 h). If starting material was not completely consumed, additional PPh 3 was added in portions (typically 0.05 eq) until TLC indicated complete turnover. This aza-ylide was used as a solution.

GSP-4 Synthesis of the ureas
The azido amine hydrochloride (1.1-1.3 eq) was dissolved or suspended in THF and DIPEA (1.2-4.0 eq) added. The mixture was added to the solution of the aza-ylide. And then argon atmosphere was exchanged to CO 2 . The mixture was stirred at room temperature or at elevated temperature overnight. The solvent was removed under a stream of nitrogen and the residue purified by F-SPE where applicable and/ or FCC.

GSP-5 Synthesis of the amides
The carboxylic acid (1.6 eq) was dissolved in CH 2 Cl 2 . Oxalyl chloride (2.4-3.6 eq) and a few drops of DMF were added sequentially. The reaction solution was stirred at room temperature until TLC indicated complete turnover (typically 2-3 h). The solvent was removed under reduced pressure and the residue dissolved in CH 2 Cl 2 or THF and was added to the solution of the aza-ylide. The mixture was stirred at room temperature overnight and quenched by the addition of MeOH. The solvent was removed under a stream of nitrogen and the residue dissolved in EtOAc. The organic phase was washed with 5% aq. HCl, sat. aq. NaHCO 3 and brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude product was purified by FCC.

GSP-6 Cu-catalysed azide alkyne cycloadditions
The linear precursor (1.0 eq) was dissolved in THF at a concentration of 1 mM and DIPEA (3.0 eq) added. Argon was bubbled through the solution for 20 min. CuI (2.0 eq) was added and the reaction mixture refluxed until analytical HPLC indicated complete turnover. The solvent was removed under a stream of nitrogen and the residue purified by FCC and/ or preparative HPLC if necessary.

GSP-7 Ru-catalysed azide-alkyne cycloadditions
The linear precursor (1.0 eq) was dissolved in THF at a concentration of 1 mM. Argon was bubbled through the solution for 20 min. [Cp*RuCl] 4 (0.1 eq) was added and the reaction mixture refluxed until analytical HPLC indicated complete turnover. The reaction solvent was removed under a stream of nitrogen and the residue purified by FCC and/ or preparative HPLC.

GSP-8 Transesterification
The fluorous-tagged macrocycle (1.0 eq) was dissolved in MeOH and 0.05 M MeONa in MeOH (1.0 eq) added. The reaction mixture was stirred at room temperature until TLC or LCMS indicated S69 complete turnover. HCl in dioxane (4 M; 1.2 eq) was added to neutralise the reaction. The solvent was removed under a stream of nitrogen and the residue purified by FCC and/ or preparative HPLC if necessary.

GSP-9 Hydrolysis
The fluorous-tagged macrocycle (1.0 eq) was dissolved in THF (20 mM) and LiOH (5.0 eq) in water (half of the THF volume) was added at 0 °C. After stirring at room temperature until TLC or LCMS indicated complete turnover (typically 2 h), the reaction was neutralised by the addition of HCl in dioxane (4 M) and stirred for another 10 min. The solvent was removed under a stream of nitrogen and the residue purified using preparative HPLC.

Hex-5-yn-1-amine hydrochloride (8a)
5-Hexyn1-ol (3.31 mL, 30.0 mmol, 1.0 eq) and pyridine (3.20 mL, 39.0 mmol, 1.3 eq) were dissolved in anhydrous CH 2 Cl 2 (75 ml) and tosyl chloride (7.44 g, 39.0 mmol, 1.3 eq) was added in portions over 5 min at 0 °C. After stirring at rt for 18 h, CH 2 Cl 2 (60 mL) was added and the organic phase was washed with 0.1 M aq. HCl (2 × 90 mL), sat. aq. NaHCO 3 (100 mL) and brine (100 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford the crude tosylate as a colourless oil (8.60 g) which was used without further purification. The crude tosylate was dissolved in DMF (60 ml) and sodium azide (2.89 g, 44.5 mmol, 1.5 eq) was added. The mixture was stirred at rt overnight. Et 2 O (40 mL) and water (20 mL) were added and the mixture was stirred for 5 min. The phases were separated and the aqueous phase was extracted with Et 2 O (3 × 40 mL). The organic phases were combined and washed with water (50 mL), sat. aq. NaHCO 3 (50 mL) and water (50 mL), dried over Na 2 SO 4 and filtered. The solution was concentrated under reduced pressure to leave 20-30 mL of solvent. The solution of crude azide was used in the next step without further purification. THF (75 ml) and PPh 3 (8.66 g, 33.0 mmol, 1.10 eq) were added to the ethereal solution of crude azide and the mixture stirred for 1.5 h. Water (1.5 mL, 83.3 mmol, 2.8 eq) was added and the solution stirred at rt for 4 days. The mixture was concentrated under reduced pressure to reduce the solvent volume by half and 3 M aq. HCl was added to adjust the pH to 1 and the aqueous mixture was washed with CH 2 Cl 2 (3 × 90 mL). Na 2 CO 3 (s) was added to the aqueous phase until pH ≥ 12, and the aqueous phase extracted with CH 2 Cl 2 (20 × 30 mL). The organic phase was dried over MgSO 4 , filtered, and 4 M HCl in dioxane (10.5 mL, 42 mmol, 1.4 equiv.) was added. The cloudy solution was concentrated under reduced pressure to afford a white solid which was triturated with Et 2 O (2 × 60 mL) to give 8a (1.0 g, 7.5 mmol, 25% over 3 steps) as a white solid. NH 2 HCl .
64a (2.4 mg, 5.7 µmol, 53%) was obtained as a white solid as the major product, and 64b (1.6 mg, 3.8 µmol, 35%) was obtained as a yellow solid as the minor product.